Auto/allo-immune defense receptors for the selective targeting of activated pathogenic t cells and nk cells

ABSTRACT

Embodiments of the disclosure concern engineered auto/allo-immune defense receptor (ADR)-expressing T cells that selectively target activated T cells, including pathogenic T cells, to incapacitate them. The chimeric receptors comprise moieties for targeting 4-1BB, OX40, and CD40L, for example, whose expression is indicative of activated T cells. In particular embodiments, there are methods of preventing or treating conditions associated with activated T cells using adoptive T-cell transfer of cells encoding the ADRs.

This application is a continuation of U.S. Non-Provisional applicationSer. No. 17/049,561 filed Oct. 21, 2020 which is a national phaseapplication under 35 U.S.C. § 371 that claims priority to InternationalApplication No. PCT/US2019/029163 filed Apr. 25, 2019, which claimspriority to U.S. Provisional Patent Application 62/662,817, filed Apr.26, 2018, all of which are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 CA126752awarded by National Institutes of Health and the National CancerInstitute. The government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing, named“Seq_Listing.txt” (18,804 bytes), created Feb. 26, 2021, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

Embodiments of the disclosure include at least the fields of immunology,cell biology, molecular biology, and medicine.

BACKGROUND

Unwanted activation of T- and NK-cells often promotes life-threateningallo-immune reactions in patients receiving transplants or thirdparty-derived therapeutic cells, leading to rejection of a transplantedorgan/tissue or development of graft-versus-host disease (GvHD).Likewise, unwanted activation of autoreactive T-cells can lead todevastating autoimmune conditions, such as diabetes mellitus, autoimmunecolitis, and multiple sclerosis. Currently, most of these diseases arenot curable because of the inability to selectively eliminate pathogenicT cells. Instead, the patients are often treated with immunosuppressivedrugs that render them immunodeficient and therefore susceptible toinfections and malignant transformations.

The present disclosure provides solutions for a long felt need in theart of safe and effective tissue transplantation and adoptive celltransfer, including utilizing off-the-shelf cells, by enhancing theirability of the transferred cells to control pathogenic conditionsbecause of unwanted activation of the immune system.

BRIEF SUMMARY

The present disclosure is directed to compositions and methods relatedto cells utilized for adoptive transfer to control pathogenic conditionsdue to immune activation. The composition and methods apply toautologous and allogeneic cells. Although some steps may be taken toreduce the reactivity of allogeneic cells in the recipient individual,such cells would still be targeted by the immune system of the recipient(primarily T- and NK-cells), which would recognize them as foreignleading to rejection and limiting therapeutic benefit.

The present disclosure overcomes this problem by modifying adoptivetherapy cells to target activated pathogenic T, NK-T, and NK cells toprevent or treat medical conditions associated with their presence. Inparticular embodiments, the compositions and methods utilize adoptiveT-cell transfer with cells that express receptors that selectivelytarget pathogenic T cells while sparing resting T cells. In specificembodiments, the adoptive T-cells for transfer are engineered to expresschimeric molecules that target pathogenic T cells that express certaintarget molecules whose presence on T cells is indicative of pathogenic Tcells. In particular embodiments the disclosure concernsauto/allo-immune defense receptors (ADRs) for the selective targeting ofpathogenic T-cells.

Particular embodiments of the disclosure include methods of protectingengineered allogeneic T cells from elimination in a host individual byproviding to the individual cells armed with ADRs. Embodiments alsoinclude methods that avoid allo-immune reactions in individualsreceiving tissue or organ transplants, for example.

In particular embodiments, cells encompassed by the disclosure have beenmodified or can be modified to allow them to survive in a recipient,including an allogeneic recipient. In specific cases, cells for adoptivecell therapy (including T cells, NKT cells, and so forth) are suitablefor being utilized “Off-the-shelf”, which herein refers to cells kept ina repository, or bank, that may be provided (with or without furthermodification) to an individual in need thereof for a specific purpose.The individual in many cases is not the individual from which the cellswere originally derived. The cells utilized in such a manner may bepre-manufactured to express an ADR, although in some cases the cells areobtained from a bank and afterwards are modified to express an ADR. Thebanked cells may or may not also express a CAR or recombinant TCR, orthe cells obtained from the bank may afterwards be modified to express aCAR or recombinant TCR. Such practices allow for ease of use of thirdparty-derived therapeutic cells without immune rejection by a host andwithout having to manufacture a patient-specific produce every time oneis needed.

In particular embodiments, there is an isolated polynucleotide,comprising sequence encoding: (1) one or more of an OX40-specificligand, a 4-1BB-specific ligand, CD40L-specific ligand, or functionalderivatives thereof; that is operably linked to (2) a signaling domainpromoting T-cell activation. The polynucleotide may comprise anOX40-specific ligand, a 4-1BB-specific ligand, or a CD40L-specificligand. The OX40-specific ligand may be OX40L, an antibody that targetsOX40, an OX40L-Fc fusion, or a combination thereof, or any otherengineered protein capable of specific binding to OX40. The4-1BB-specific ligand may be 4-1BBL, an antibody that targets 4-1BB, a4-1BBL-Fc fusion, or a combination thereof, or any other engineeredprotein capable of specific binding to 4-1BB. The CD40L-specific ligandmay be CD40, an antibody that targets CD40L, a CD40-Fc fusion, or anyother engineered protein capable of specific binding to CD40L orcombination thereof. In at least certain cases, the polynucleotidefurther comprises sequence that encodes a spacer between (1) and (2),such as between 10 and 220 amino acids in length, for example. Thespacer may have sequence that facilitates surface detection with anantibody, such as the spacer being detectable with an anti-Fc Ab. Thespacer may comprise an IgG Fc portion.

In particular embodiments, polynucleotides of the disclosure may furtherencode a chimeric antigen receptor, a T-cell receptor, or both. Thepolynucleotide may be in any form including present on a vector, such asa viral vector (retroviral vector, lentiviral vector, adenoviral vector,or adeno-associated viral vector) or non-viral vector (plasmid,transposon, etc.). In particular cases, the polynucleotide is present ina cell, including a eukaryotic cell or a bacterial cell. The cell may bean immune cell, such as a T cell. The cell may be engineered. The cellmay comprise one or more chimeric antigen receptors (CARs) and/or one ormore engineered T cell receptors (TCRs).

Polypeptides expressed by any polynucleotide encompassed by thedisclosure are included as part of the disclosure. In particularembodiments there is a polypeptide, comprising: (1) one or more of anOX40-specific ligand, a 4-1BB-specific ligand, and CD40; that isoperably linked to (2) a signaling domain promoting T-cell activation.The signaling domain promoting T-cell activation may be from CD3 zetasubunit, DAP12, an Fc receptor, or a combination thereof.

Any cell encompassed by the disclosure is part of the disclosure. Inspecific embodiments, any chimeric receptor-expressing cell is part ofthe disclosure, including cells, comprising any polynucleotidecontemplated herein and/or any polypeptides contemplated herein. Thecell may be an engineered cell. The cell may be an immune cell, such asa T cell, including a CAR-transduced T cell and/or a T cell receptor(TCR)-transduced T cell. In specific embodiments, the cell is engineeredto lack endogenous expression of one or more genes, such as lack one ormore of 4-1BB, OX40 and/or CD40L. The cell may be engineered usingCRISPR/Cas9, zinc finger nucleases, TALE nucleases, or meganucleases.Alternatively, the cell may be engineered to prevent surface expressionof ADR ligands, for example, by trapping the ADR ligand with a specificantibody or a receptor anchored in the endoplasmic reticulum or anotherintracellular compartment.

In one embodiment there is a method of avoiding rejection of allogeneiccells, tissue, or organs in an individual, comprising the step ofdelivering to the individual an effective amount of allogeneic immunecells expressing an engineered chimeric receptor that comprises anextracellular domain that targets compounds that are selectively presenton activated T cells and that comprises CD3 zeta, wherein the deliveringstep results in the following in the individual: (1) inhibition ofendogenous alloreactive T cells in the individual; and/or (2)suppression of NK cell activation in the individual. In specificembodiments, the allogeneic cells are the allogeneic immune cellsexpressing the chimeric receptor. The allogeneic cells may express achimeric antigen receptor and/or an engineered T cell receptor. Theallogeneic immune cells may be delivered to the individual before,during, and/or after tissue and/or organ transplantation in theindividual. In specific cases, the activated T cells are pathogenic Tcells.

In one embodiment, there is a method of selectively targeting activatedT cells in an individual, comprising the step of providing to theindividual an effective amount of cells expressing an engineeredchimeric receptor, said chimeric receptor comprising: (1) anextracellular domain that targets compounds that are selectively presenton activated T cells; and (2) a signaling domain promoting T-cellactivation. The signaling domain promoting T-cell activation may bederived from CD3 zeta subunit, DAP12, an Fc receptor, anyITAM-comprising sequence, or a combination thereof. In specific cases,the activated T cells are pathogenic T cells.

In a certain embodiment, there is a method of preventing or treating amedical condition related to activated T cells in an individual,comprising the step of delivering to the individual an effective amountof immune cells expressing an engineered chimeric receptor thatselectively targets said activated T cells, said chimeric receptorcomprising: (1) an extracellular domain that targets compounds that areselectively present on activated T cells; and (2) a signaling domainpromoting T-cell activation. The medical condition may be an autoimmunedisorder, such as graft rejection, graft-versus-host disease, type Idiabetes, multiple sclerosis, autoimmune colitis, or a combinationthereof, for example.

In one embodiment, there is a method of avoiding NK cell-mediated hostrejection of allogeneic T cells, tissues, or organs in an individual,comprising the step of providing to the individual an effective amountof immune cells expressing an engineered chimeric receptor thatcomprises an extracellular domain that targets compounds that areselectively present on activated T cells and that also comprises asignaling domain promoting T-cell activation. The immune cellsexpressing the engineered chimeric receptor are the allogeneic T cells,in certain cases. The immune cells may express a chimeric antigenreceptor and/or an engineered T cell receptor. The amount of immunecells expressing the engineered chimeric receptor that are provided tothe individual may be in a range of 10²-10¹² per m². The cellsexpressing the chimeric receptor may be provided to the individualsystemically or locally. The immune cells may be T cells. The immunecells may be delivered to the individual once or more than once.

The foregoing has outlined rather broadly the features and technicaladvantages of the present disclosure in order that the detaileddescription that follows may be better understood. Additional featuresand advantages will be described hereinafter which form the subject ofthe claims herein. It should be appreciated by those skilled in the artthat the conception and specific embodiments disclosed may be readilyutilized as a basis for modifying or designing other structures forcarrying out the same purposes of the present designs. It should also berealized by those skilled in the art that such equivalent constructionsdo not depart from the spirit and scope as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe designs disclosed herein, both as to the organization and method ofoperation, together with further objects and advantages will be betterunderstood from the following description when considered in connectionwith the accompanying figures. It is to be expressly understood,however, that each of the figures is provided for the purpose ofillustration and description only and is not intended as a definition ofthe limits of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure, referenceis now made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIGS. 1A-1E. ADRs can be expressed on cell surface of immune cells andpromote cytotoxicity against respective targets. (FIG. 1A) Schematic ofADR. GFP is optional (FIG. 1B) Expression of ADR on the cell surface(FIG. 1C) Expansion of ADR T cells after transduction (FIG. 1D)Cytotoxicity of ADR T cells against target cells expressing ADR ligands(FIG. 1E) Expansion of wild-type vs 4-1BB KO T cells expressing 4-1BBADR and their cytotoxicity against 4-1BB+ targets showing that knockingout ADR ligand on T cells can further enhance expansion and cytotoxicityand demonstrating co-expression of ADR and its ligand on T cells is notrequired for ADR-T cell expansion or function

FIGS. 2A-2E. Selective expression of ADR ligands on activated T cellsenables their selective elimination by ADR T cells. (FIG. 2A-FIG. 2C)Expression of ADR ligands on resting vs activated T cells after TCRstimulation. (FIG. 2D) Absence of cytotoxicity of ADR T cells againstresting CD4+ and CD8+ T cells (FIG. 2E) Elimination of activated CD4+and CD8+ T cells by ADR T cells after a 48 h coculture.

FIGS. 3A-3F. Expression of 4-1BB ADR protects T cells from immunerejection in an MLR model (FIG. 3A) Representative dot plots showingTCRKO T cells xo-expressing ADR are protected from rejection aftercoculture with allogeneic PBMC at a 1:10 ADR T:PBMC ratio. (FIG. 3B-FIG.3C) Absolute counts of donor T cells and allogeneic T cells in the PBMCduring coculture (FIG. 3D-FIG. 3F) same for virus-specific ADR T cells.

FIGS. 4A-4C. Expression of ADR protects allogeneic virus-specific Tcells from immune rejection in a mixed lymphocyte reaction in vitro(FIG. 4A) Representative dot plots showing ADR VST are protected fromimmune rejection by recipient allogeneic PBMC (FIG. 4B-FIG. 4C) Absolutecounts of recipient T cells and donor VST at various time points duringMLR.

FIG. 5. ADR VSTs retain anti-viral function. ADR VSTs were coculturedwith viral pepmix-pulsed monocytes, and monocyte counts indicated thatthey eliminated viral infected cells equally well compared to unmodifiedVSTs.

FIGS. 6A-6H. Activated NK cells upregulate ADR ligands and can beselectively targeted by ADR T cells (FIG. 6A-FIG. 6B) Expression of4-1BB on resting vs activated NK cells (FIG. 6C) Residual counts ofresting vs activated NK cells after 24 hr coculture with of 4-1BB ADR Tcells (FIG. 6D) ADR T cells lacking MHC are protected from immunerejection by allogeneic PBMC by controlling the expansion of NK cells(FIG. 6E) Absolute counts of donor T cells and allogeneic NK cellsduring coculture. (FIG. 6F) ADR T cells lacking MHC resist immunerejection by NK cells upon 48 h coculture at a 1:1 E:T ratio. (FIG.6G-FIG. 6H) ADR T cells control the expansion of alloreactive NK cellsduring MLR with PBMC, with absolute counts of NK cells plotted in H.

FIGS. 7A-7E. ADR expression protects allogeneic T cells from immunerejection in vivo (FIG. 7A) Schematic of the mouse model of immunerejection where mice were given T cells from an HLA-A2+ donor after asublethal irradiation, followed by administration of allogeneic HLA-A2−T cells 4 days later. (FIG. 7B-FIG. 7C) Control T cells from theHLA-A2-donor were rejected by Day 18 while ADR-expressing cells wereprotected (FIG. 7C) Absolute counts of T cells from HLA-A2+ and HLA-A2−donors at various time points. (FIG. 7D) Modified in vivo model whereinstead of allogeneic T cells mice received whole PBMC (containing bothT- and NK-cells) from donor 1. (FIG. 7E) Representative flow plotsshowing ADR T cells were protected from immune rejection and alsoprotected mice from rapid onset of fatal GvHD.

FIGS. 8A-8E. Coexpression of CAR and ADR preserves functions of bothreceptors (FIG. 8A) Schematic representation of an immune cellco-expressing ADR and a CAR (FIG. 8B) Coexpression of CAR and ADR on thecell surface (FIG. 8C) Cytotoxicity of CAR-ADR T cells against NALM-6(CD19+ CAR target) (FIG. 8D) Cytotoxicity of CAR-ADR T cells againstactivated T cells (ADR target) (FIG. 8E) Cytotoxic activity of CAR-ADR Tcells against both targets upon simultaneous co-culture with both celltargets.

FIGS. 9A-9E. CAR-ADR T cells are protected from immune rejection andexert potent anti-tumor activity (FIG. 9A) Schematic of the mouse model.Mice received allogeneic T cells from Donor 1 and b2mKO NALM6 24 hrapart, followed by a single dose of CAR-ADR T cells from Donor 2. (FIG.9B) Kinetics of T cells from Donor 2 in peripheral blood (FIG. 9C)Kinetics of Donor 1 T cells in the experimental groups (FIG. 9D)Leukemia burden in mice (FIG. 9E) overall survival of mice.

FIGS. 10A-10C. CAR-ADR T cells are protected from immune rejection andexert potent anti-tumor activity in a solid tumor model. (FIG. 10A)Schematic of the mouse model. Mice received allogeneic T cells fromDonor 1 and b2mKO neuroblastoma cell line CHLA255 24 hr apart, followedby a single dose of CAR-ADR T cells from Donor 2. (FIG. 10B) Donor 2 GD2CAR T cells were rejected by D18, whereas CAR-ADR T cells resistedallogeneic rejection and persisted in peripheral blood. (FIG. 10C) Tumorburden in mice, * indicates xenogeneic-GvHD associated deaths in ATC+GD2CAR T group.

FIGS. 11A-11E. TCR-knockout CAR-ADR T cells are protected from immunerejection and exert potent anti-tumor activity (FIG. 11A) Schematic ofthe mouse model. Mice received allogeneic T cells from Donor 1 and b2mKONALM6 24 hr apart, followed by a single dose of TCR-edited CAR-ADR Tcells from Donor 2. (FIG. 11B) Kinetics of T cells from Donor 2 inperipheral blood (FIG. 11C) Kinetics of Donor 1 T cells in theexperimental groups (FIG. 11D) Leukemia burden in mice (FIG. 11E)overall survival of mice.

FIGS. 12A-12D. ADR T cells protect mice against fatal xenogeneic GvHD(FIG. 12A) Schematic of the model (FIG. 12B) Expansion of FFLuc-labeledADR T cells in vivo (FIG. 12C) Kinetics of weight gain/loss in mice(FIG. 12D) Overall survival of mice.

FIGS. 13A-13G. 2nd generation ADR with CD28 intracellular signalingdomain (ADR.28zeta). (FIG. 13A) Structure of ADR.28zeta. (FIG. 13B-FIG.13C) in vitro cytotoxicity of ADR.28zeta against target-expressing celllines. (FIG. 13D-FIG. 13G) ADR.28zeta protected mice from xeno-GvHD.(FIG. 13D) Schematic of the model (FIG. 13E) Expansion of FFLuc-labeledADR.28zeta T cells in vivo (FIG. 13F) Kinetics of weight gain/loss inmice (FIG. 13G) Overall survival of mice.

FIG. 14. Cytotoxicity of ADR-expressing cells in cancer. (left)Cytotoxicity of 4-1BB ADR-expressing T cells against HDLM-2 Hodgkin'slymphoma cells (right) Cytotoxicity of 4-1BB ADR-expressing T cellsagainst K562 chronic myeloid leukemia (CML) cells. Absolute counts oftumor cells upon a 48 h coculture at a 1:1 effector-to-target ratio isshown.

DETAILED DESCRIPTION

As used herein, the words “a” and “an” when used in the presentspecification in concert with the word comprising, including the claims,denote “one or more.” Some embodiments of the invention may consist ofor consist essentially of one or more elements, method steps, and/ormethods of the invention. It is contemplated that any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

The term “subject,” as used herein, generally refers to an individual inneed of a therapy for a medical condition of any kind. A subject can bean animal of any kind. The subject can be any organism or animal subjectthat is an object of a method or material, including mammals, e.g.,humans, laboratory animals (e.g., primates, rats, mice, rabbits),livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens),household pets (e.g., dogs, cats, and rodents), horses, and transgenicnon-human animals. The subject can be a patient, e.g., have or besuspected of having a disease (that may be referred to as a medicalcondition), such as one or more infectious diseases, one or more geneticdisorders, one or more cancers, or any combination thereof. The diseasemay be pathogenic. The subject may being undergoing or having undergoneantibiotic treatment. The subject may be asymptomatic. The subject maybe healthy individuals. The term “individual” may be usedinterchangeably, in at least some cases. The “subject” or “individual”,as used herein, may or may not be housed in a medical facility and maybe treated as an outpatient of a medical facility. The individual may bereceiving one or more medical compositions via the internet. Anindividual may comprise any age of a human or non-human animal andtherefore includes both adult and juveniles (i.e., children) and infantsand includes in utero individuals. The individual may be of any race andgender. It is not intended that the term connote a need for medicaltreatment, therefore, an individual may voluntarily or involuntarily bepart of experimentation whether clinical or in support of basic sciencestudies.

The term “engineered” as used herein refers to a molecule that is notpresent in nature and has been generated by the hand of man, such as bygenetic recombination techniques standard in the art.

In the context of the present disclosure, an “effective amount” or a“therapeutically effective amount” refers to the amount of cells that,when administered to an individual, allows for targeting of activated Tcells and/or alleviates the signs and or symptoms of a medical conditionor prevents a medical condition. The actual amount to be administeredcan be determined based on studies done either in vitro or in vivo wherethe functional immune cells exhibit pharmacological activity against amedical condition.

I. Auto/Allo-Immune Defense Receptors and Compositions and Uses Thereof

The present disclosure encompasses synthetic chimeric receptor moleculesthat provide for selective targeting of activated T cells, includingpathogenic T cells. The engineered molecules are synthetic and may beproduced by recombinant technology. The molecules may be referred to asauto/allo-immune defense receptors that target activated T cells,including activated pathogenic T cells, including with high specificity.

In particular embodiments, auto/allo-immune defense receptors (ADRs)comprise an entity that targets one or more compounds that areupregulated on activated T cells. Although the compound that isupregulated on activated T cells may be any one or combination thereof,in specific embodiments OX40, 4-1BB, and CD40L are upregulated onactivated T cells and are the subjects of which the ADRs are targeting.The ADRs are present on allogeneic immune cells that are allogeneic withrespect to the individual receiving the cells, in particularembodiments. In other instances, the ADRs are expressed on autologous Tcells, xenogeneic cells, and/or synthetic cells.

A. Auto/Allo-Immune Defense Receptor (ADR) Molecules

ADR molecules are synthetic, non-natural, and produced by the hand ofman and comprise at least (1) an extracellular domain that targetscompounds that are selectively present on activated T cells (and inspecific embodiments, the extracellular domain is a protein orfunctional fragment or derivative thereof that targets one or morecompounds that are upregulated on activated T cells); that is operablylinked to (2) a signaling domain promoting T-cell activation, includingthose derived from CD3 zeta subunit, DAP12, and Fc receptors, or anotherITAM-comprising sequence, for example. The ADR molecule may comprise orconsist of or consist essentially of elements (1) and (2). In at leastcertain cases, the ADR comprises one or more components of a Type Itransmembrane protein and/or one or more components of a Type IItransmembrane protein.

In specific embodiments, in the ADR molecule the extracellular domaincomprises a protein that selectively binds an associated protein on anactivated T cell. For example, the ADR extracellular domain may comprisea ligand for a receptor on an activated T cell, or the ADR extracellulardomain may comprise a receptor for a ligand on an activated T cell.

In specific embodiments, in the ADR molecule the extracellular domaincomprises a ligand for OX40, a ligand for 4-1BB, and/or CD40. Theseparticular examples have associated proteins on activated T cells thatare OX40, 4-1BB, and CD40L, respectively. In alternative embodiments,other particular compositions on the activated T cells are targeted. Forexample, one may target other activation markers that are upregulated onthe cell surface of T cells (like CD69, CD25, CD71, etc.) can betargeted using a similar approach. In such cases, the corresponding ADRmolecule would have a respective CD69, CD25, or CD71 ligand, or anantibody-derived targeting moiety, instead of 4-1BB/OX40-specificligands.

In some cases, activated T cells are targeted that have upregulation ofexpression of OX40, in contrast to T cells that are not activated. Totarget these activated T cells, a ligand of OX40 could be utilized inthe ADR to be able to target the activated T cells. In cases wherein aligand for OX40 is utilized in the ADR, the ligand for OX40 may be anysuitable ligand for OX40 including at least OX40L, an antibody (orfunctional fragment thereof) that binds to OX40, a fusion of Fc withOX40L, or functional derivatives or fragments thereof. OX40L may also bereferred to as tumor necrosis factor (ligand) superfamily, member 4(tax-transcriptionally activated glycoprotein 1, 34 kDa), OX40L, CD252,TNFSF4, TXGP1, OX-40L, or gp34.

In some cases, activated T cells are targeted that have upregulation ofexpression of 4-1BB, in contrast to T cells that are not activated. Totarget these activated T cells, a ligand of 4-1BB could be utilized inthe ADR to be able to target the activated T cells. In cases wherein aligand for 4-1BB is utilized in the ADR, the ligand for 4-1BB may be anysuitable ligand for 4-1BB including 4-1BBL, an antibody (or functionalfragment thereof) that targets 4-1BB, a fusion of Fc with 4-1BBL, orfunctional derivatives or fragments thereof.

In certain cases, activated T cells are targeted that have upregulationof expression of CD40L, in contrast to T cells that are not activated.To target these activated T cells, a receptor for CD40L could beutilized in the ADR to be able to target the activated T cells. In caseswherein a receptor for CD40L is utilized in the ADR, the ADR maycomprise CD40 (that may also be referred to as Bp50, CDW40, TNFRSF5, orp50), an antibody (or functional fragment thereof) that targets CD40L,or functional derivatives or fragments thereof.

In some cases, the ADR molecule comprises two or more extracellulardomains to facilitate targeting of the activated T cells. Suchcombinations may enhance targeting of activated T cells generally or mayallow for specific targeting of certain subsets of activated T cells.For example, the ADR may comprise both OX40L and 4-1BBL as extracellulardomains in the same ADR molecule to allow for targeting of activated Tcells that express either OX40 or 4-1BB. Such an example of acombination would selectively target activated T cells that expresseither OX40 or 4-1BB regardless of whether or not those activated Tcells also express CD40L. Analogously, ADRs may comprise both CD40 andOX40L to target activated T cells that express either CD40L or OX40regardless of whether or not those activated T cells also express 4-1BB.

In the ADR molecule, the extracellular domain may be operably linked toone or more components, including components that are part of the ADRmolecule. One such component may be a protein that mediates downstreamsignaling during T cell activation. In particular embodiments the ADRcomprises CD3zeta (also referred to as CD247, CD3-ZETA, CD3H, CD3Q,CD3Z, IMD25, T3Z, or TCRZ) or a functional fragment or derivativethereof. CD3zeta mediates downstream ITAM-derived signaling during Tcell activation. Other ITAM-containing signaling domains may includethose derived from DAP12, Fc receptors, other CD3 subunits, etc. Thesignaling domains may be non-covalently linked to the ADR via anotherdomain.

In particular embodiments, the ADR comprises a spacer between the CD3zeta and the extracellular protein that targets one or more compoundsthat are upregulated on activated T cells. In other cases, a spacer isnot utilized. The spacer may comprise sequence that is inert orcontributes substantially little or nothing with respect to any functionthe ADR may have, whereas in other cases the spacer comprises sequencethat enhances a function of the ADR and/or allows it to be detectableand/or able to be targeted for inhibition, as examples. In specificembodiments, the spacer comprises encoded protein sequence thatfacilitates detection of cells that express the ADR. For example, thespacer may encode Fc region or fragments thereof that would allow forsurface detection of the cells, such as using anti-Fc Abs. In particularembodiments, the spacer provides separation between the ligand bindingdomain and the membrane to avoid potential steric hindrances, such asthose caused by the splicing of Type II transmembrane proteins (4-1BBL,OX40L) with the Type I ADR backbone (TM, signaling domains). The spacermay be of any suitable length, including about 10-220 amino acids as anexample. The spacer length may be in a range of 10-220, 10-200, 10-150,10-100, 10-50, 25-200, 25-150, 25-100, 25-75, 25-50, 50-200, 50-150,50-125, 50-100, 50-75, 75-200, 75-150, 75-100, 100-200, 100-175,100-150, 100-125, 125-200, 125-175, 125-150, 150-200, 150-175, 175-200,and so forth. The spacer may be about 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220amino acids in length. In other cases, the spacer is less than 10 aminoacids or more than 200 amino acids.

In some cases, ADRs comprise one, two, three, or more costimulatorydomains that enhance cytokine production from the cells that express theADR. The costimulatory domains may be derived from the intracellularsignaling domains of costimulatory proteins including CD28, CD27, 4-1BB,OX40, ICOS, CD30, HVEM, CD40, and so forth. As an example only, when theADR comprises 4-1BBL, the costimulatory domain of the ADR may or may notbe from 4-1BB.

In some embodiments, the ADRs will comprise a transmembrane domain thatmay be of any kind so long as it allows the CD3 zeta component of theADR to be located intracellularly and the extracellular domain thattargets one or more compounds that are upregulated on activated T cellsto be located extracellularly. In other instances, ADRs are solubleproteins that can bind to the respective ligand on activated T cells andpromote cytotoxicity by crosslinking TCR (e.g., ADR-CD3 T-cell engagerprotein). In a case wherein the extracellular domain is from a surfaceprotein having a transmembrane domain, (CD40, for example), the ADR maycomprise the transmembrane domain from that corresponding endogenousmolecule. In some cases in which the ADR molecule comprises one or morecostimulatory domains, the transmembrane domain (TM) may be from thesame endogenous molecule that has the costimulatory domain. Examples ofTMs include those from CD3, CD8α, CD27, CD28, 4-1BB, OX40, CD4, etc.

In an example of a ADR polypeptide, the components may be in aparticular N-terminal (N) to C-terminal (C) order. For a general ADR,the receptor may comprise one of the following (as examples only) andwherein the extracellular domain comprises the protein that selectivelybinds an associated protein on an activated T cell:

N-extracellular domain-signaling domain-C

N-extracellular domain-CD3zeta-C

N-extracellular domain-spacer-CD3zeta-C

N-extracellular domain-spacer-costimulatory domain-CD3zeta-C

N-extracellular domain-spacer-two costimulatory domains-CD3zeta-C

N-two extracellular domains-spacer-costimulatory domain-CD3zeta-C

N-two extracellular domains-spacer-two costimulatory domains-CD3zeta-C

In any case, the transmembrane domain may be C-terminal with respect tothe spacer. A signal peptide at the N-terminus may be utilized tofacilitate expression of Type II ligands (such as OX40L and 4-1BBL) on aType I transmembrane protein backbone (such as the transmembrane domain,signaling domains, CD3 zeta).

In some cases, the ADR comprises one or more detectable markers, such asmarkers that are colorimetric, fluorescent, and/or radioactive, and soforth. Examples include green fluorescent protein, blue fluorescentprotein, and so forth.

The ADR may be in the form of a polynucleotide or polypeptide expressedby a polynucleotide, although the ADR may be synthetically generated asa protein. Recombinant technology to produce ADR polynucleotides andpolypeptides are known in the art.

In certain cases, a ADR polynucleotide is in an expression construct oris part of an expression construct present on a vector that may be aviral vector or a non-viral vector. Examples of non-viral vectorsinclude plasmids. Examples of viral vectors include lentiviral,retroviral, adenoviral, and adeno-associated viral vectors. Any vectorexpressing the ADR will have appropriate element(s) to allow expressionin an eukaryotic cell, including an immune cell, such as a T cell, NKcell, or NKT cell, for example. Such appropriate elements includepromoters and so forth.

4-1BB ADR (SEQ ID NO: 1)MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFV TAAGITLGMDELYKOX40 ADR (SEQ ID NO: 2)MEFGLSWLFLVAILKGVQCQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSHLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIHKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK CD40L ADR (SEQ ID NO: 3)MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRESKYGPPCPPCPGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSAAAGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE FVTAAGITLGMDELYK

In certain embodiments, the extracellular domain that targets activatedT cells comprises an antibody or functional fragment or derivativethereof. The term “antibody,” as used herein, refers to animmunoglobulin molecule that specifically binds with an antigen.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions of intactimmunoglobulins. Antibodies are typically tetramers of immunoglobulinmolecules. The antibodies in the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chainantibodies and humanized antibodies (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

In some cases, the extracellular domain of the ADR comprises an antibodyfragment. The term “antibody fragment” refers to a portion of an intactantibody and refers to the antigenic determining variable regions of anintact antibody. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,scFv antibodies, and multispecific antibodies formed from antibodyfragments.

Synthetic antibodies may be used in the ADR. By the term “syntheticantibody” as used herein, is meant an antibody which is generated usingrecombinant DNA technology, such as, for example, an antibody expressedby a bacteriophage as described herein. The term should also beconstrued to mean an antibody that has been generated by the synthesisof a DNA molecule encoding the antibody and which DNA molecule expressesan antibody protein, or an amino acid sequence specifying the antibody,wherein the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology that is available and well knownin the art.

B. Cells Expressing ADRs

Allogeneic cells used for adoptive transfer are prone to having limitedefficacy because of the immune reaction by the recipient individual.Although in some cases, the cells may be modified to remove endogenousTCR (such as with CRISPR), for example to prevent graft-versus-hostdisease, alternatively one can utilize virus-specific T cells (intact orCAR/TCR-modified) to retain anti-viral activity, which is useful incertain pathologic conditions. Although such VSTs have very limitedgraft-versus-host activity because their TCRs are more restricted toviral antigens, they are still susceptible to deleterious reaction bythe recipient.

Encompassed in the disclosure are cells that are improved for allogeneicuse by being modified to express a synthetic ADR molecule. Thus, thedisclosure includes cells harboring the ADR as a polynucleotide and asan expressed ADR polypeptide on the surface of the cells. In particularcases, the ADR-expressing cells are produced for the purpose of beingmaintained in a repository for off-the-shelf use. The cells may behoused in a repository already being configured to express an ADR, orthey may be housed in a bank and configured to express an ADR followingretrieval from the repository. Certain cells, such as bacterial cells,may be utilized to generate the ADR molecules, whereas other cells, suchas eukaryotic cells, harboring the ADR may be used for methods of thedisclosure including targeting activated T cells. As shown herein,ADR-expressing immune cells selectively eliminate activated T cells andADR-expressing immune cells are protected against cytolysis byalloreactive T cells.

Cells that express the ADR molecule may be of any kind, but in specificembodiments they are immune cells, for example immune effector cells,such as T cells, NK cells, NKT cells, or cell lines derived from thesaid lineages or engineered to have cytotoxic activity that have beenmodified to express the ADR and are therefore not found in nature.Populations of the non-natural ADR-expressing cells are contemplated,including populations that are at least 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% of the population being ADR-expressingcells. The cells may be generated by standard methods of transfection ortransduction of synthetic ADR polynucleotides, as an example.

In some cases, the ADR molecules that modified to express the ADRmolecule may already be engineered or subsequently are engineered tohave another engineered, non-natural molecule other than the ADR. Forexample, cells that express chimeric antigen receptors (CAR) orengineered T cell receptors (TCR) and in doing so can protect such cellsagainst host rejection and therefore increase their therapeutic potency.Cells expressing one or more CARs and/or one or more TCRs may beengineered to express one or more ADRs, or cells that express one ormore ADRs may be engineered to express one or more CARs and/or one ormore TCRs. Thus, in some cases a ADR is expressed on a different vectorthan a CAR and/or TCR, yet in other cases a ADR molecule is expressed onthe same vector as a CAR and/or TCR. In cases wherein a ADR is expressedon the same vector as a CAR (as an example), the ADR and CAR expressionmay be directed from the same or different regulatory elements. In anycase, the ADR and CAR may be expressed as a single polypeptide with acleavable element between them, such as 2A.

In cases wherein ADR-expressing cells also express a CAR or TCR, the CARor TCR may target any particular antigen. In cases wherein CARs areemployed, the CARs may be first generation, second generation, thirdgeneration and so on. The CAR may be bispecific, in specific cases.

In some cases, cells expressing the ADR molecules are engineered, suchas engineered to lack expression of one or more endogenous molecules. Inspecific cases the cells are engineered to lack expression of one ormore endogenous genes that would facilitate fratricide of the cellsotherwise. In specific cases the cells expressing the ADR molecules areengineered to lack expression of 4-1BB or OX40, for example. Engineeringof the cells may occur by CRISPR/Cas9, merely as an example.

II. Methods of Using Auto/Allo-Immune Defense Receptors

Embodiments of the disclosure include methods of providing an effectiveamount of ADR-expressing cells to an individual for any purpose. Methodsinclude providing selectively targeting activated T cells in anindividual for any purpose. The activated T cells are targeted byexposing activated T cells to an effective amount of ADR-expressingimmune cells, such as ADR-expressing T cells. Such exposure may have oneor more resultant applications, for example.

In some embodiments, the ADRs are used to selectively target activatedimmune cells other than activated T cells, such as B cells (that wouldbe useful for controlling unwanted B-cell responses such as with lupus,rheumatoid arthritis, etc.), as well as targeting activation of innateimmunity (such as macrophage activation syndrome, and so on). In otherembodiments, the ADRs are utilized to specifically target malignantcells expressing their corresponding target, including 4-1BB or OX40 orCD40L, for example.

The regimen for providing to an individual an effective amount ofADR-expressing cells may be known or determined by an individual orindividuals delivering the cells for therapy or prevention, orregardless of the method of use. In preventative cases, for example, thecells may be delivered prior to detection of one or more symptoms, orthe cells may be delivered following detection of one or more symptomsbut before further symptom(s) develop and/or worsen. For treatmentcases, the individual may be provided the effective amount of cellsafter one, two, or more symptoms develop and may be after clinicaldiagnosis.

In specific aspects of the methods, the individual may be given a singledose of a therapeutically effective amount of cells, or the individualmay be given multiple deliveries of the therapeutically effective amountof the cells, such as multiple deliveries separated by 1, 2, 3, 4, 5, 6,7 days, or 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months, or 1, 2, 3, 4, 5, or more years, or any range there between,for example. The time between doses may vary in a single regimen.

The administration of the ADR-expressing cells may be via any suitableroute to the individual, including locally or systemically. In specificembodiments the ADR-expressing cells are delivered intravenously,orally, rectally, topically, intramuscularly, by infusion, enterically,nasally, by inhalation, sublingually, bucally, transdermally,subcutaneously, and so forth. The cells may or may not be delivered as abolus. With multiple administrations, the cells may or may not beprovided to the individual in different delivery routes. When the cellsare delivered to an individual, they may be delivered in apharmaceutically acceptable carrier or excipient. Particular examples ofdoses for ADR-expressing cells include 10⁴ cells/m², 10⁵ cells/m², 10⁶cells/m², 10⁷ cells/m², 10⁸ cells/m², 10⁹ cells/m², 10¹⁰ cells/m², 10¹¹cells/m², or 10¹² cells/m² and ranges there between.

A. Use for Off-the-Shelf Embodiments

The disclosure encompasses cells for adoptive transfer that are able tobe utilized off-the-shelf, including able to be obtained from arepository for the purpose of use in an individual other than theindividual from which the cells were originally obtained. The cells mayalready express the ADR prior to being deposited in the repository, orthe cells may be modified afterwards to express the ADR. The cells maybe any kind of immune effector cells for adoptive transfer. The cellsprior to deposit in the repository or after obtaining them from therepository may be modified to express a tumor-specific receptor, forexample (e.g., a CAR or a TCR).

As shown elsewhere herein, cells expressing ADRs selectively targetactivated T- and NK-cells while sparing resting subsets. The ADRsprotect allogeneic T cells from immune rejection mediated by T- and/orNK-cells in vitro in addition to protecting T-cells from immunerejection in vivo. The ADRs in doing so do not interfere with thefunction of an engineered anti-tumor receptor (CAR, as an example) asT-cells co-expressing ADR and CAR can efficiently eliminate both tumorand activated T cells in vitro. The disclosure further providesanti-cancer activity in vivo in a mouse model using “off-the-shelf” Tcells co-expressing CAR and ADR while retaining resistance to immunerejection from allogeneic T-cells present in the same mice. As anexample, in FIG. 14 it is shown that 4-1BB ADR is effective against4-1BB+ tumor cells such that ADR can be used as a therapeutic modalityagainst 4-1BB-expressing malignancies.

In particular embodiments, “off-the-shelf” therapeutic cells express ADRto resist immune rejection and either retain endogenous TCR specificity(e.g., to viral or tumor antigens) or have endogenous TCRs replaced withengineered anti-tumor receptors, such as one or more CARs and/or one ormore recombinant TCRs.

In specific embodiments, off-the-shelf cells are housed in a repositoryand may be modified for a specific purpose either before or afterdeposit in the repository. For example, ADR-expressing T cells may behoused in a repository and ready for use, such as after a tissue ororgan transplant, to prevent graft rejection. ADR-expressing T cells maybe housed in a repository and may be selected or engineered with anative or transgenic TCR, for example against viral infection or cancer.ADR-expressing T cells may be housed in a repository and may betransduced with a CAR directed to cancer or pathogenic infection.ADR-expressing cells may be housed in a repository and may be transducedwith one or more CARs and/or one or more TCRs directed to specificcancer-associated antigens or neoantigens expressed by the patient'sspecific tumor.

In some cases, banked allogeneic cells are utilized for prevention ofrejection of solid organ grafts by destroying rejecting host immunecells, particularly in cases when the ADR-expressing cells were notthemselves alloreactive.

Although the cells that are housed in the repository may be allogeneicwith respect to a recipient individual, in alternative embodiments thecells housed in the repository are autologous with respect to arecipient individual. For example, an individual with cancer may haveADR-expressing T cells deposited in a repository for subsequent use,such as in the event that the cancer comes out of remission. In othercases, autologous ADR-expressing cells are housed in a repository fortreatment of autoimmune disorders.

B. Use in Autoimmune Disorders

Unwanted activation of endogenous autoreactive T cells in an individualcan lead to devastating autoimmune diseases for the individual, such asdiabetes mellitus, autoimmune colitis, and multiple sclerosis. Inparticular embodiments, one or more autoimmune disorders are preventedor treated using ADR-expressing immune cells in the individual thatimpacts the autoimmune disorder (or its potential development) byinhibiting endogenous autoreactive T cells in the individual. Inspecific embodiments, such use of the ADR-expressing cells sparesresting non-pathogenic naïve and memory T cells in the individual. Thus,certain methods of the disclosure utilize particular cells modified toexpress ADRs that are provided to an individual in a sufficient amountto target activated T cells, including pathogenic T cells, therebyinitiating destruction of the activated T cells.

In vivo activation of T cells with unwanted specificity may causepathogenicity, and in particular embodiments cells expressing one ormore ADRs target the activated T cells. In some cases, ADR-expressingcells target pathogenic cells that are a subset of activated T cells.

In particular cases, ADR-expressing T cells can be used to prevent orreverse life-threatening and debilitating conditions driven by activatedT cells (organ rejection, graft-versus-host disease, type I diabetes,multiple sclerosis, autoimmune colitis, lupus, rheumatoid arthritis, asexamples) using adoptive T-cell transfer with the ADR-expressing Tcells.

In some cases, the ADR-expressing T cells also comprise one or morecompositions other than the ADR that facilitate treatment or preventionof one or more autoimmune disorders.

In some cases, an individual being provided the ADR-expressing T cellsis given one or more additional therapies to prevent or treat one ormore autoimmune disorders. The individual may or may not be given one ormore immunosuppressive drugs, for example, such as glucocorticoids,cytostatics, antibodies, and/or drugs acting on immunophilins. Inaddition or alternatively, the individual may be given one or moreappropriate vaccines.

In some cases, an individual is at risk for an autoimmune disorder andis provided an effective amount of ADR-expressing cells to prevent onsetof the autoimmune disorder or to delay onset and/or lessen one or moresymptoms, including in severity and/or duration, for example. Anindividual at risk for an autoimmune disorder, for example, is onehaving a personal or family history, being a female of certainethnicity, and so forth. The individual may have one autoimmune disorderand desires to prevent or reduce the severity and/or duration or delaythe onset of another autoimmune disorder(s), in some cases.

Examples of autoimmune disorders that may be prevented or treated withADR-expressing cells include at least the following: Achalasia;Addison's disease; Adult Still's disease; Agammaglobulinemia; Alopeciaareata; Amyloidosis; Ankylosing spondylitis; Anti-GBM/Anti-TBMnephritis; Antiphospholipid syndrome; Autoimmune angioedema; Autoimmunedysautonomia; Autoimmune encephalomyelitis; Autoimmune hepatitis;Autoimmune inner ear disease (AIED); Autoimmune myocarditis; Autoimmuneoophoritis; Autoimmune orchitis; Autoimmune pancreatitis; Autoimmuneretinopathy; Autoimmune urticarial; Axonal & neuronal neuropathy (AMAN);Balò disease; Behcet's disease; Benign mucosal pemphigoid; Bullouspemphigoid; Castleman disease (CD); Celiac disease; Chagas disease;Chronic inflammatory demyelinating polyneuropathy (CIDP); Chronicrecurrent multifocal osteomyelitis (CRMO); Churg-Strauss Syndrome (CSS)or Eosinophilic Granulomatosis (EGPA); Cicatricial pemphigoid; Cogan'ssyndrome; Cold agglutinin disease; Congenital heart block; Coxsackiemyocarditis; CREST syndrome; Crohn's disease; Dermatitis herpetiformis;Dermatomyositis; Devic's disease (neuromyelitis optica); Discoid lupus;Dressler's syndrome; Endometriosis; Eosinophilic esophagitis (EoE);Eosinophilic fasciitis; Erythema nodosum; Essential mixedcryoglobulinemia; Evans syndrome; Fibromyalgia; Fibrosing alveolitis;Giant cell arteritis (temporal arteritis); Giant cell myocarditis;Glomerulonephritis; Goodpasture's syndrome; Granulomatosis withPolyangiitis; Graves' disease; Guillain-Barre syndrome; Hashimoto'sthyroiditis; Hemolytic anemia; Henoch-Schonlein purpura (HSP); Herpesgestationis or pemphigoid gestationis (PG); Hidradenitis Suppurativa(HS) (Acne Inversa); Hypogammalglobulinemia; IgA Nephropathy;IgG4-related sclerosing disease; Immune thrombocytopenic purpura (ITP);Inclusion body myositis (IBM); Interstitial cystitis (IC); Juvenilearthritis; Juvenile diabetes (Type 1 diabetes); Juvenile myositis (JM);Kawasaki disease; Lambert-Eaton syndrome; Leukocytoclastic vasculitis;Lichen planus; Lichen sclerosus; Ligneous conjunctivitis; Linear IgAdisease (LAD); Lupus; Lyme disease chronic; Meniere's disease;Microscopic polyangiitis (MPA); Mixed connective tissue disease (MCTD);Mooren's ulcer; Mucha-Habermann disease; Multifocal Motor Neuropathy(MMN) or MMNCB; Multiple sclerosis; Myasthenia gravis; Myositis;Narcolepsy; Neonatal Lupus; Neuromyelitis optica; Neutropenia; Ocularcicatricial pemphigoid; Optic neuritis; Palindromic rheumatism (PR);PANDAS; Paraneoplastic cerebellar degeneration (PCD); Paroxysmalnocturnal hemoglobinuria (PNH); Parry Romberg syndrome; Pars planitis(peripheral uveitis); Parsonnage-Turner syndrome; Pemphigus; Peripheralneuropathy; Perivenous encephalomyelitis; Pernicious anemia (PA); POEMSsyndrome; Polyarteritis nodosa; Polyglandular syndromes type I, II, III;Polymyalgia rheumatic; Polymyositis; Postmyocardial infarction syndrome;Postpericardiotomy syndrome; Primary biliary cirrhosis; Primarysclerosing cholangitis; Progesterone dermatitis; Psoriasis; Psoriaticarthritis; Pure red cell aplasia (PRCA); Pyoderma gangrenosum; Raynaud'sphenomenon; Reactive Arthritis; Reflex sympathetic dystrophy; Relapsingpolychondritis; Restless legs syndrome (RLS); Retroperitoneal fibrosis;Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt syndrome;Scleritis; Scleroderma; Sjögren's syndrome; Sperm & testicularautoimmunity; Stiff person syndrome (SPS); Subacute bacterialendocarditis (SBE); Susac's syndrome; Sympathetic ophthalmia (SO);Takayasu's arteritis; Temporal arteritis/Giant cell arteritis;Thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); Transversemyelitis; Type 1 diabetes; Ulcerative colitis (UC); Undifferentiatedconnective tissue disease (UCTD); Uveitis; Vasculitis; Vitiligo;Vogt-Koyanagi-Harada Disease; and Wegener's granulomatosis (orGranulomatosis with Polyangiitis (GPA)).

C. Use to Eliminate NK Cells

ADR-expressing immune cells may be utilized to eliminate NK cells incases wherein it is desirable to do so. As demonstrated herein, thepresence of ADRs on certain immune cells provides a specific cytotoxicactivity against NK cells that are involved with mediating rapidrejection of HLA^(low) or HLA-mismatched cells. Thus, in situationswhere it is desirable to maintain HLA^(low) or HLA-mismatched cells, forexample to be able to utilize adoptive transfer of allogeneic cells intocertain individuals, use of ADR-expressing cells avoids NK cellactivation and rejection of the HLA-mismatched cells. Specifically, asshown herein, co-culture of T cells expressing ADRs leads to eliminationof NK cells and thus offsets the NK cell-mediated host rejection ofADR-expressing allogeneic T cells.

D. Use to Facilitate the Engraftment of Allogeneic Cell/Tissue/OrganTransplant

In a specific aspect of avoiding activation of alloreactive T cells, onemay avoid rejection of allogeneic cells, tissues, or organs inindividuals receiving transplants, which is mainly mediated by apopulation of alloreactive T cells from the recipient. Activation ofrecipient's alloreactive T cells would lead to graft failure, forexample, if steps were not taken to avoid such rejection. Therefore, inparticular embodiments, methods of transplanting cells, tissue, ororgans into an individual utilize delivery of an effective amount ofADR-expressing cells before, during, and/or after the respectivetransplants of cells, tissue(s), or organ(s). In some cases, theADR-expressing cells are not themselves part of the cells, tissue(s), ororgan(s) that are the subject of the transplant, whereas in other casesthe ADR-expressing cells are part of the respective cells, tissue(s), ororgan(s).

Tissue for transplantation may be of any kind including at least skin,cornea, bone, tendons, heart valves, veins, or arteries, for example.Organ for transplantation may be of any kind, including heart, kidneys,liver, lungs, pancreas, intestine, and thymus, for example.

In particular embodiments, ADR-expressing cells enhance allogeneic celluse in an individual as a two-pronged approach: (1) they inhibit theendogenous alloreactive T-cells in the individual; and (2) they suppressNK cell-mediated rejection in the individual. As such, the ADR moleculescan enhance the persistence and activity of any type of thirdparty-derived therapeutic cells in the individual including, forexample, allogeneic therapeutic cells, including T-cells, NK cells, NK-Tcells, mucosal associated invariant T cells (MATT) and other cytotoxiccells, including those expressing engineered constructs such as chimericantigen receptor (CAR), transgenic TCR, etc.

E. Use in Prophylaxis or Treatment of Graft-Versus-Host Disease (GvHD)During Allogeneic Cell/Tissue/Organ Transplant

In another specific aspect of avoiding activation of alloreactive Tcells, one may avoid life-threatening allo-immune reactions inindividuals receiving transplants of allogeneic cells, tissues, ororgans. Such transplants would contain donor alloreactive T cells thatwould elicit development of graft-versus-host disease (GvHD), forexample, if steps were not taken to avoid their activation. Therefore,in particular embodiments, methods of transplanting cells, tissue, ororgans into an individual utilize delivery of an effective amount ofADR-expressing cells before, during, and/or after the respectivetransplants of cells, tissue(s), or organ(s). In some cases, theADR-expressing cells are not themselves part of the cells, tissue(s), ororgan(s) that are the subject of the transplant, whereas in other casesthe ADR-expressing cells are part of the respective cells, tissue(s), ororgan(s).

Tissue for transplantation may be of any kind including at least skin,cornea, bone, tendons, heart valves, veins, or arteries, for example.Organ for transplantation may be of any kind, including heart, kidneys,liver, lungs, pancreas, intestine, and thymus, for example.

III. Production of ADR-Expressing Cells

Cells expressing the ADR molecules may be produced in a variety of ways,all of which may be routine in the art. The production methods mayinclude obtaining the cells to be modified to express the ADR moleculeand also include generation of the ADR molecules.

A. Sources of T Cells

Prior to expansion and genetic modification of the ADR-expressing Tcells of the disclosure, a source of T cells may be obtained from asubject. Such a step of obtaining may or may not be part of the method.In some cases, obtaining T cells to be modified and their manipulationmay be performed by a party other than the party that provides the ADRexpressing-T cells to an individual. T cells can be obtained from anumber of sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present disclosure, any number of T celllines available in the art may be used. In certain embodiments, T cellscan be obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficoll™separation. In one embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets, for example. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment, the cells are washed with phosphate buffered saline(PBS). In an alternative embodiment, the wash solution lacks calcium andmay lack magnesium or may lack many if not all divalent cations. Asthose of ordinary skill in the art would readily appreciate a washingstep may be accomplished by methods known to those in the art, such asby using a semi-automated “flow-through” centrifuge (for example, theCobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics CellSaver 5) according to the manufacturer's instructions. After washing,the cells may be resuspended in a variety of biocompatible buffers, suchas, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyre-suspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. In another embodiment, it may bedesirable to use lower concentrations of cells. By significantlydiluting the mixture of T cells and surface (e.g., particles such asbeads), interactions between the particles and cells is minimized

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.Many freezing solutions and parameters are known in the art. In certainembodiments, cryopreserved cells are thawed and washed as describedherein and allowed to rest for one hour at room temperature prior toactivation using the methods of the present invention.

Also contemplated in the context of the disclosure is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773,1993). In a further embodiment, the cells are isolated for a patient andfrozen for later use in conjunction with (e.g., before, simultaneouslyor following) bone marrow or stem cell transplantation, T cell ablativetherapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cells are isolatedprior to and can be frozen for later use for treatment following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan.

B. Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressthe ADR, the T cells can be activated and expanded generally usingmethods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005. Generally, the T cells of the disclosure are expandedby contact with a surface having attached thereto an agent thatstimulates a CD3/TCR complex associated signal and a ligand thatstimulates a co-stimulatory molecule on the surface of the T cells. Suchprocesses are known in the art. In other instances, T cells can bemodified to express ADR without prior activation.

C. Generation of ADR Molecules

Turning generally to polynucleotides that encode the ADR, the nucleicacid sequences coding for the ADR molecules can be obtained usingrecombinant methods known in the art, such as, for example by screeninglibraries from cells expressing the gene, by deriving the gene from avector known to include the same, or by isolating directly from cellsand tissues containing the same, using standard techniques.Alternatively, the ADR polynucleotide of interest can be producedsynthetically, rather than cloned.

In brief summary, the expression of synthetic polynucleotides encodingADRs is typically achieved by operably linking a nucleic acid encodingthe ADR polypeptide or portions thereof to a promoter, and incorporatingthe construct into an expression vector. The vectors can be suitable forreplication and integration eukaryotes. Typical cloning vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the desired nucleicacid sequence.

The ADR polynucleotide can be cloned into a number of types of vectors.For example, the nucleic acid can be cloned into a vector including, butnot limited to a plasmid, a phagemid, a phage derivative, an animalvirus, and a cosmid. Vectors of particular interest include expressionvectors, replication vectors, probe generation vectors, and sequencingvectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral-based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1alpha (EF-1alpha). However, other constitutive promoter sequences mayalso be used, including, but not limited to the simian virus 40 (SV40)early promoter, mouse mammary tumor virus (MMTV), human immunodeficiencyvirus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, anavian leukemia virus promoter, an Epstein-Barr virus immediate earlypromoter, a Rous sarcoma virus promoter, as well as human gene promoterssuch as, but not limited to, the actin promoter, the myosin promoter,the hemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of an ADR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing ADR polynucleotides into a cellare known in the art. In the context of an expression vector, the vectorcan be readily introduced into a host cell, e.g., mammalian, bacterial,yeast, or insect cell by any method in the art. For example, theexpression vector can be transferred into a host cell by physical,chemical, or biological means.

Physical methods for introducing a ADR polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). One method for the introduction of a polynucleotide into ahost cell is calcium phosphate transfection.

Biological methods for introducing a ADR polynucleotide of interest intoa host cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20.degree. C. Chloroform is used as the onlysolvent since it is more readily evaporated than methanol. “Liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

In some instances, the ADR molecules may be integrated into anendogenous nucleic acid of the cells. One may have a target site forhomologous recombination, where it is desired that a construct beintegrated at a particular locus. For example,) can knock-out anendogenous gene and replace it (at the same locus or elsewhere) with thegene encoded for by the construct using materials and methods as areknown in the art for homologous recombination. For homologousrecombination, one may use either OMEGA or 0-vectors. CRISPR/Cas9, zincfinger nucleases, TALE nucleases, meganucleases, and other site directednucleases may be used to target and cleave a specific site in the genometo promote homologous recombination.

The exemplary T cells that have been engineered to include theADR-expressing construct(s) may be grown in culture under selectiveconditions and cells that are selected as having the construct may thenbe expanded and further analyzed, using, for example; the polymerasechain reaction for determining the presence of the construct in the hostcells. Once the engineered host cells have been identified, they maythen be used as planned, e.g. expanded in culture or introduced into ahost organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, in a wide variety of ways. The cells maybe introduced at the site of the tumor, in specific embodiments,although in alternative embodiments the cells home to the cancer or aremodified to home to the infected tissue. The number of cells that areemployed will depend upon a number of circumstances, the purpose for theintroduction, the lifetime of the cells, the protocol to be used, forexample, the number of administrations, the ability of the cells tomultiply, the stability of the recombinant construct, and the like. Thecells may be applied as a dispersion, generally being injected at ornear the site of interest. The cells may be in aphysiologically-acceptable medium.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

EXAMPLES

The following examples are presented in order to more fully illustrateparticular embodiments of the disclosure. They should in no way,however, be construed as limiting the broad scope of the disclosure.

Example 1 Auto/Allo-Immune Defense Receptors for the Selective Targetingof Pathogenic T Cells

Disclosed herein is a novel approach to specifically target pathogenic Tcells using auto/allo-immune defense receptors (ADRs) expressed onnormal T cells. ADR-expressing T cells find and eliminate only activatedT cells and spare resting non-pathogenic naïve and memory T cells, whichconstitute the majority of circulating lymphocytes

The concept of the ADR-mediated targeting is based on the observationthat within 24 h of activation, T cells transiently upregulatecostimulatory genes 4-1BB, OX40, and/or CD40L on their cell surface. Theexpression of 4-1BB, OX40, and/or CD40L is only maintained when theT-cells are actively cytotoxic and is gradually downregulated within 4-5days, when TCR signaling has stopped. Notably, activated CD8⁺ T cellsshowed higher magnitude of 4-1BB expression whereas CD4⁺ T cellspreferentially expressed OX40 and/or CD40L. Apart from activated Tcells, ADR ligands may only be expressed on activated NK cells and onsome other non-critical and replenishable subsets of cells. The patternof expression of 4-1BB, OX40, and CD40L makes these genes attractivetargets to target activated cells with high specificity while avoidingpermanently damaging critical immune and non-immune tissues.

To explore the feasibility of targeting these activated T cells,auto/allo-immune defense receptors (ADRs) were designed to comprise of a4-1BB- or OX40-specific ligand, or a CD40L-specific receptor directlyconnected via a spacer to a CD3ζ chain encoded in a gammaretroviralvector SFG. The spacer region was incorporated a) to enable integrationof type II proteins 4-1BBL and OX40L into the Type I backbone of the ADRand b) to facilitate detection of ADR on the cell surface by FACSstaining. Transduction of T cells with this construct efficiently forcedADR expression on the cell surface. These ADR T cells had potent androbust cytotoxicity against 4-1BB-, OX40-, and CD40L-expressing cells,eliminating 90-99% of the target cells within 48 h. These resultsdemonstrate the feasibility of generating functional 4-1BB-, OX40-, andCD40L-specific ADR T cells.

Because ADR signaling in T cells in turn upregulates 4-1BB, OX40, andCD40L, thus promoting fratricide and impeding effector cell expansion,the effects of CRISPR/Cas9 genomic disruption of ADR target genes in theeffector T cells were explored. The inventors had previously shown thatthis CRISPR/Cas9 approach can prevent fratricide of primary human Tcells expressing a CD7-specific CAR. In this context, with CRISPR/Cas9the inventors were able to knock out 4-1BB expression in ˜70% of ADR Tcells that consistently increased ADR T cell expansion >2-fold at 48 hfollowing coculture with 4-1BB⁺ target cells without affecting thecytotoxicity.

Next, the ability of ADR T cells was tested to selectively eliminateactivated T cells. 4-1BB, OX40, and CD40L ADR T cells were co-culturedwith fluorescently labeled resting or CD3/CD28 activated T cells.Residual live CD4⁺ and CD8⁺ T cells were quantified by flow cytometrywith counting beads. There was no reactivity against resting autologousT cells after 72h of co-culture with T cells expressing 4-1BB-, OX40-,or CD40L-specific ADR (FIG. 2B). In contrast, co-culture of 4-1BB ADR Tcells with CD3/CD28-activated T cells eliminated most CD8⁺ and some CD4⁺T cells within 48 h. Incubation with OX40 ADR T cells resulted in areciprocal high-level depletion of activated CD4⁺ T cells and modestdepletion of activated CD8+ T cells. CD40L ADR T cells produced moderatecytotoxic effect on activated CD4⁺ T cells yet no effect was seen onCD8⁺ T cells. The differential targeting profiles of OX40, CD40L and4-1BB ADR T cells against activated CD4⁺ and CD8⁺ T cells correlateswith observed differences in the magnitude and kinetics of OX40, CD40Land 4-1BB expression on each T cell subset. This property of ADRs can beutilized to preferentially target either or both subsets of allo- orauto-reactive T cells according to need. Therefore, ADR expressionenables T cells to specifically target activated (pathogenic) T cellsbut spare resting cells, suggesting their clinical use.

It was assessed whether virus-specific T cells (VST) expressing ADRs canresist allogeneic rejection in an in vitro mixed lymphocyte reaction(MLR) assay. CMV-specific T cells were generated from an HLA-A2-negativedonor and mixed control non-transduced or ADR-transduced VST withalloreactive HLA-A2⁺ PBMC at a 1:2 cell-to-cell ratio. The inventorsthen cultured the cells for 12 days. At the end of co-culture, controlVSTs were almost completely eliminated by HLA-A2⁺ PBMC whereas VSTsexpressing either 4-1BB ADR or OX40 ADR resisted rejection. Takentogether, these results demonstrate the feasibility and selectivity oftargeting activated T cells using the newly developed ADR platformembodiment.

Example 2 Auto/Allo-Immune Defense Receptors for the Selective Targetingof NK Cells

The ADRs demonstrate a specific cytotoxic activity against NK cells, akey cell population mediating rapid rejection of HLA^(low) orHLA-mismatched cells.

NK cells are capable of recognizing HLA-mismatched cells or cells withlow HLA expression, as a part of the anti-tumor and anti-viral immunesurveillance. Adoptive transfer of allogeneic cells into immunorepletepatients would thus result in NK-cell activation and rejection of theHLA-mismatched cells. Here, it is shown that co-culture of T cellsexpressing 4-1BB- and OX40-specific auto/allo-immune defense receptors(ADRs) leads to elimination of NK cells and thus would offset the NKcell-mediated host rejection of ADR-armed allogeneic T cells. Therefore,ADRs do not only inhibit the alloreactive T-cell response but alsosuppress NK cell mediated rejection, further supporting the applicationof ADRs to enhance the persistence and activity of “off-the-shelf”therapeutic T cells.

Example 3 ADR-Expressing T Cells Eliminate Target Cells

ADRs can be expressed on cell surface of immune cells and promotecytotoxicity against respective targets. FIG. 1A illustrates one exampleof a schematic of ADR (a label such as GFP is optional). The expressionof ADR on the surface of T cells was confirmed (FIG. 1B) and the cellswere expanded commensurate with controls (FIG. 1C). (FIG. 1D) TheADR-expressing T cells were cytotoxicity against target cells expressingcorresponding ADR ligands (FIG. 1D). FIG. 1E demonstrates expansion ofwild-type vs 4-1BB KO T cells expressing 4-1BB ADR and theircytotoxicity against 4-1BB+ targets. Knocking out the ADR ligand on Tcells can further enhance expansion and cytotoxicity, and co-expressionof ADR and its ligand on T cells is not required for ADR-T cellexpansion or function (FIG. 1E).

Selective expression of ADR ligands on activated T cells enables theirselective elimination by ADR T cells. Expression of ADR ligands onresting vs activated T cells after TCR stimulation is determined (FIG.2A-FIG. 2C). There was no cytotoxicity of ADR T cells against restingCD4+ and CD8+ T cells (FIG. 2D), yet there was elimination of activatedCD4+ and CD8+ T cells by ADR T cells after a 48 h co-culture (FIG. 2E).

As one example, expression of 4-1BB ADR protects T cells from immunerejection in an MLR model. Representative dot plots showing TCRKO Tcells co-expressing ADR are protected from rejection after co-culturewith allogeneic PBMC at a 1:10 ADR T:PBMC ratio (FIG. 3A). Absolutecounts of donor T cells and allogeneic T cells in the PBMC duringco-culture (FIG. 3B-FIG. 3C) are the same for virus-specific ADR T cells(FIG. 3D-FIG. 3F).

Expression of ADR protects allogeneic virus-specific T cells from immunerejection in a mixed lymphocyte reaction in vitro. Representative dotplots showing ADR VST are protected from immune rejection by recipientallogeneic PBMC (FIG. 4A). The absolute counts of recipient T cells anddonor VST at various time points during MLR are provided in FIG. 4B andFIG. 4C.

In FIG. 5, ADR VSTs retain anti-viral function. ADR VSTs wereco-cultured with viral pepmix-pulsed monocytes, and monocyte countsindicated that they eliminated viral infected cells equally wellcompared to unmodified VSTs.

Activated NK cells upregulate ADR ligands and can be selectivelytargeted by ADR T cells. Expression of 4-1BB on resting vs activated NKcells is confirmed (FIG. 6A-FIG. 6B). Residual counts of resting vsactivated NK cells after 24 hr co-culture with 4-1BB ADR T cells aredetermined (FIG. 6C). In FIG. 6D, ADR T cells lacking MHC are protectedfrom immune rejection by allogeneic PBMC by controlling the expansion ofNK cells. Absolute counts of donor T cells and allogeneic NK cellsduring co-culture are determined in FIG. 6E. ADR T cells lacking MHCresist immune rejection by NK cells upon 48 h co-culture at a 1:1 E:Tratio (FIG. 6F). ADR T cells control the expansion of alloreactive NKcells during MLR with PBMC (FIG. 6G), with absolute counts of NK cellsplotted in FIG. 6H.

ADR expression protects allogeneic T cells from immune rejection invivo. In FIG. 7A, one example is shown of a mouse model of immunerejection where mice were given T cells from an HLA-A2+ donor after asublethal irradiation, followed by administration of allogeneic HLA-A2−T cells 4 days later. Control T cells from the HLA-A2− donor wererejected by Day 18 while ADR-expressing cells were protected (FIG. 7B).Absolute counts of T cells from HLA-A2+ and HLA-A2− donors at varioustime points were determined (FIG. 7C). A modified in vivo model in FIG.7D depicts where, instead of allogeneic T cells mice, received wholePBMC (containing both T- and NK-cells) from donor 1. Representative flowplots in FIG. 7E show that ADR T cells were protected from immunerejection and also protected mice from rapid onset of fatal GvHD.

Coexpression of CAR and ADR preserves functions of both receptors. FIG.8A illustrates an example of a representation of an immune cellco-expressing ADR and a CAR Coexpression of a CAR and an ADR on the cellsurface were confirmed (FIG. 8B). In FIG. 8C, cytotoxicity is shown ofCAR-ADR T cells against NALM-6 (A CD19+ CAR target), as one example of atarget. Cytotoxicity of the CAR-ADR T cells against activated T cells(ADR target) were also determined in FIG. 8D. Cytotoxic activity ofCAR-ADR T cells against both targets upon simultaneous co-culture withboth cell targets is demonstrated in FIG. 8E.

CAR-ADR T cells are protected from immune rejection and exert potentanti-tumor activity. An example of a mouse model and a regimen isdepicted in FIG. 9A. Mice received allogeneic T cells from Donor 1 andb2mKO NALM6 24 hr apart, followed by a single dose of CAR-ADR T cellsfrom Donor 2, as one example of a regimen. Kinetics of T cells fromDonor 2 in peripheral blood are provided in FIG. 9B, and kinetics ofDonor 1 T cells in the experimental groups are provided in FIG. 9C. FIG.9D shows leukemia burden in the mice, with determination of overallsurvival of the mice (FIG. 9E).

FIGS. 13A-13C. CAR-ADR T cells are protected from immune rejection andexert potent anti-tumor activity in a solid tumor model. Schematic of anexample of a mouse model and treatment is shown in FIG. 10A, whereinmice received allogeneic T cells from Donor 1 and b2mKO neuroblastomacell line CHLA255 24 hr apart, followed by a single dose of CAR-ADR Tcells from Donor 2. Donor 2 GD2 CAR T cells were rejected by D18,whereas CAR-ADR T cells resisted allogeneic rejection and persisted inperipheral blood (FIG. 10B). Tumor burden in mice is shown in FIG. 13C,where * indicates xenogeneic-GvHD associated deaths in ATC+GD2 CAR Tgroup.

TCR-knockout CAR-ADR T cells are protected from immune rejection andexert potent anti-tumor activity. Schematic of the mouse model isprovided in which mice received allogeneic T cells from Donor 1 andb2mKO NALM6 24 hr apart, followed by a single dose of TCR-edited CAR-ADRT cells from Donor 2 (FIG. 11A). Kinetics of T cells from Donor 2 inperipheral blood are provided (FIG. 11B). Kinetics of Donor 1 T cells inthe experimental groups are shown (FIG. 11C). Leukemia burden in mice(FIG. 11D) and overall survival of mice are provided (FIG. 11E).

ADR T cells protect mice against fatal xenogeneic GvHD. Schematic of amodel is provided in FIG. 12A, and expansion of FFLuc-labeled ADR Tcells in vivo is demonstrated (FIG. 12B). Kinetics of weight gain/lossin mice were determined (FIG. 12C). Overall survival of mice is depicted(FIG. 12D).

Second generation ADR with CD28 intracellular signaling domain(“ADR.28zeta”)(as one example) were utilized. One example of a structureof ADR.28zeta is depicted (FIG. 13A). In vitro cytotoxicity wasdetermined of ADR.28zeta against target-expressing cells ((FIG. 13B andFIG. 13C). ADR.28zeta protected mice from xeno-GvHD lines (FIG. 13B).Schematic of the model (FIG. 13D) is shown. Expansion of FFLuc-labeledADR.28zeta T cells in vivo was confirmed (FIG. 13E) Kinetics of weightgain/loss in mice (FIG. 13F), and the overall survival of mice wasdetermined (FIG. 13G).

Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the design as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thepresent disclosure, processes, machines, manufacture, compositions ofmatter, means, methods, or steps, presently existing or later to bedeveloped that perform substantially the same function or achievesubstantially the same result as the corresponding embodiments describedherein may be utilized according to the present disclosure. Accordingly,the appended claims are intended to include within their scope suchprocesses, machines, manufacture, compositions of matter, means,methods, or steps.

1. An isolated polynucleotide, comprising sequence encoding apolypeptide, wherein said polypeptide comprises: (1) one or more of anOX40-specific ligand, a 4-1BB-specific ligand, CD40L-specific ligand, orfunctional derivatives thereof; that is operably linked to (2) asignaling domain promoting T-cell activation.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. The polynucleotide of claim 1, wherein theOX40-specific ligand is OX40L, an antibody that targets OX40, anOX40L-Fc fusion, or a combination thereof.
 6. The polynucleotide ofclaim 1, wherein the 4-1BB-specific ligand is 4-1BBL, an antibody thattargets 4-1BB, a 4-1BBL-Fc fusion, or a combination thereof.
 7. Thepolynucleotide of claim 1, wherein the CD40L-specific ligand is CD40, anantibody that targets CD40L, a CD40-Fc fusion, or any other engineeredprotein capable of specific binding to CD40L.
 8. (canceled)
 9. Thepolynucleotide of claim 1, wherein the polynucleotide further comprisessequence that encodes a spacer between (1) and (2).
 10. Thepolynucleotide of claim 9, wherein the spacer is between 10 and 220amino acids in length.
 11. The polynucleotide of claim 10, wherein thespacer has sequence that facilitates surface detection with an antibody.12. The polynucleotide of claim 11, wherein the spacer is detectablewith an anti-Fc Ab.
 13. The polynucleotide of claim 12, wherein thespacer comprises IgG Fc portion.
 14. The polynucleotide of claim 1,wherein the polynucleotide further encodes a chimeric antigen receptor,a T-cell receptor, or both.
 15. The polynucleotide of claim 14, whereinthere is a 2A element or IRES element on the polynucleotide betweensequence that encodes the (a) a polypeptide that comprises (1) one ormore of an OX40-specific ligand, a 4-1BB-specific ligand, CD40L-specificligand, or functional derivatives thereof; that is operably linked to(2) a signaling domain promoting T-cell activation and (b) thepolynucleotide that encodes the chimeric antigen receptor, a T-cellreceptor, or both.
 16. (canceled)
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. The polynucleotide of claim 1, wherein the polynucleotideis present in a cell.
 21. (canceled)
 22. The polynucleotide of claim 20,wherein the cell is a T cell that comprises one or more chimeric antigenreceptors or one or more engineered T cell receptors (TCRs). 23.(canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. A polypeptide expressed by a polynucleotide of claim
 1. 29. Apolypeptide, comprising: (1) one or more of an OX40-specific ligand, a4-1BB-specific ligand, and CD40; that is operably linked to (2) asignaling domain promoting T-cell activation.
 30. (canceled) 31.(canceled)
 32. A chimeric receptor-expressing cell, comprising thepolynucleotide of claim
 1. 33. The cell of claim 32, wherein the cell iscell a CAR-transduced T cell or a T cell receptor (TCR)-transduced Tcell.
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled) 38.The cell of claim 32, wherein the cell is engineered to lack endogenousexpression of one or more genes.
 39. The cell of claim 38, wherein thecell is engineered to lack endogenous expression of 4-1BB, OX40 and/orCD40L.
 40. (canceled)
 41. (canceled)
 42. A method of preparing cells forcell therapy, comprising the step of transfecting immune effector cellswith the polynucleotide of claim
 1. 43. (canceled)
 44. The method ofclaim 42, further comprising the step of modifying the cells to expressone or more chimeric antigen receptors and/or one or more recombinantT-cell receptors.
 45. The method of claim 42, further comprising thestep of providing an effective amount of the cells to an individual inneed thereof.
 46. (canceled)
 47. A method of treating an individual fora medical condition with allogeneic therapeutic cells, comprising thestep of providing to the individual a therapeutically effective amountof allogeneic cells, wherein said cells express a polypeptide comprising(1) one or more of an OX40-specific ligand, a 4-1BB-specific ligand,CD40L-specific ligand, or functional derivatives thereof; that isoperably linked to (2) a signaling domain promoting T-cell activation.48. (canceled)
 49. The method of claim 47, wherein the cells express oneor more chimeric antigen receptors and/or one or more recombinant T-cellreceptors.
 50. A method of avoiding rejection of allogeneic cells,tissue, or organs in an individual, comprising the step of delivering tothe individual an effective amount of allogeneic immune cells expressingan engineered chimeric receptor that comprises an extracellular domainthat targets compounds that are selectively present on activated T cellsand that comprises CD3 zeta, wherein the delivering step results in thefollowing in the individual: (1) inhibition of endogenous alloreactive Tcells in the individual; and/or (2) suppression of NK cell activation inthe individual.
 51. The method of claim 50, wherein the allogeneic cellsare the allogeneic immune cells expressing the chimeric receptor. 52.The method of claim 50, wherein the allogeneic cells express a chimericantigen receptor or an engineered T cell receptor.
 53. The method ofclaim 50, wherein the allogeneic immune cells are delivered to theindividual before, during, and/or after tissue and/or organtransplantation in the individual.
 54. The method of claim 50, whereinthe activated T cells are pathogenic T cells.
 55. A method ofselectively targeting activated T cells in an individual, comprising thestep of providing to the individual an effective amount of immune cellsexpressing an engineered chimeric receptor, said chimeric receptorcomprising: (1) an extracellular domain that targets compounds that areselectively present on activated T cells; and (2) a signaling domainpromoting T-cell activation.
 56. (canceled)
 57. The method of claim 55,wherein the activated T cells are pathogenic T cells.
 58. A method ofpreventing or treating a medical condition related to activated T cellsin an individual, comprising the step of delivering to the individual aneffective amount of immune cells expressing an engineered chimericreceptor that selectively targets said activated T cells, said chimericreceptor comprising: (1) an extracellular domain that targets compoundsthat are selectively present on activated T cells; and (2) a signalingdomain promoting T-cell activation.
 59. (canceled)
 60. The method ofclaim 58, wherein the medical condition is an autoimmune disorder. 61.The method of claim 58, wherein the medical condition comprises graftrejection, graft-versus-host disease, type I diabetes, multiplesclerosis, autoimmune colitis, or a combination thereof.
 62. A method ofavoiding NK cell-mediated host rejection of allogeneic T cells, tissues,or organs in an individual, comprising the step of providing to theindividual an effective amount of immune cells expressing an engineeredchimeric receptor that comprises an extracellular domain that targetscompounds that are selectively present on activated T cells and thatalso comprises a signaling domain promoting T-cell activation.
 63. Themethod of claim 62, wherein the immune cells expressing the engineeredchimeric receptor are the allogeneic T cells.
 64. The method of claim62, wherein the immune cells express a chimeric antigen receptor or anengineered T cell receptor.
 65. (canceled)
 66. (canceled)
 67. (canceled)68. (canceled)